In recent years, as a method for detecting a mutation or a polymorphism in a gene, a method for analyzing a melting curve of a double-stranded nucleic acid composed of a target nucleic acid and a probe, a melting curve analysis method, has been adopted widely. According to the melting curve analysis method, by analyzing the presence or absence of a peak at a melting temperature (Tm) of the double strand in a melting curve, the determination of a polymorphism (genotype) in a gene or the detection of the presence or absence of a mutation in a gene becomes possible.
A Tm generally is defined as below. The absorbance at 260 nm increases as a solution containing a double-stranded nucleic acid is heated. This increase is caused by the fact that the hydrogen bond between both the strands in a double-stranded nucleic acid is released by heating, and the double-stranded nucleic acid is dissociated into single-stranded nucleic acids (melting of a double-stranded nucleic acid). When every double-stranded nucleic acid is dissociated into single-stranded nucleic acids, the solution exhibits an absorbance about 1.5 times as large as the absorbance at the time when the heating was initiated (the absorbance of the solution containing only the double-stranded nucleic acid), whereby it can be determined that the melting is completed. Based on this phenomenon, a melting temperature Tm (° C.) generally is defined as a temperature at the time when the amount of increase in absorbance reaches 50% of the total amount of increase in absorbance.
By utilizing this nature of a double-stranded nucleic acid, a polymorphism or a mutation in a target site can be detected in the following manner, for example. That is, it can be achieved by the method in which, using a mutant-type detection probe that is complementary to a target nucleic acid sequence containing a mutant-type target site, a double stranded nucleic acid composed of a single-stranded nucleic acid to be analyzed and the probe is formed, the formed double-stranded nucleic acid is heat-treated, the dissociation of the double strand with temperature increase is detected by measuring signal values such as absorbance and the like, and the presence or absence of a mutation in the target site is determined by the behavior of the signal at a Tm value based on the detection result (ref. Non Patent Citation 1 and Patent Citation 1). Thus, the Tm value becomes higher as the homology of a double-stranded nucleic acid becomes higher and becomes lower as the homology of a double-stranded nucleic acid becomes lower. Thus, as evaluation criteria, Tm values of a double-stranded DNA composed of a target nucleic acid sequence with a mutant-type target site and a mutant-type detection probe that is 100% complementary to the target nucleic acid sequence and a Tm value of a double-stranded DNA composed of a nucleic acid sequence with a wild-type target site and the mutant-type detection probe are determined previously. Since the Tm value becomes higher as the homology of a double-stranded nucleic acid becomes higher as described above, the former, i.e., the Tm value in the case where the target site is of mutant type (hereinafter, also referred to as “Tmm value”) is relatively high and the latter, i.e., the Tm value in the case where the target site is of wild type (hereinafter, also referred to as “Tmw value”) is relatively low. Subsequently, a melting curve of the double-stranded nucleic acid composed of the single-stranded nucleic acid to be analyzed and the mutant-type detection probe is prepared, and whether a peak is present at the previously determined Tmm value or at the previously determined Tmw value is checked. When the peak is present at around the Tmm value, the nucleic acid sequence is a 100% match to the mutant-type detection probe, whereby the single-stranded nucleic acid to be analyzed can be determined as having a mutant-type polymorphism. On the other hand, when the peak is present at around the Tmw value, the nucleic acid sequence is a mismatch to the mutant-type detection probe in a single base, whereby the single-stranded nucleic acid to be analyzed can be determined as having a wild-type polymorphism. Further, whether the polymorphism is homozygous or heterozygous also can be determined. That is, in an analysis of a pair of alleles, when peaks are present at both around the Tmm value and around the Tmw value, the polymorphism can be determined as being heterozygous. On the other hand, when a peak is present at only around the Tmm value, the polymorphism can be determined as being a mutant-type homozygous, and when a peak is present at only around the Tmw value, the polymorphism can be determined as being a wild-type homozygous.
To conduct such melting curve analysis, usually, an amplification treatment of the target nucleic acid sequence is conducted with respect to a sample containing RNA or DNA, and the obtained amplification product is subjected to double strand formation with the probe and the disassociation by heating as described above. However, when the target nucleic acid sequence cannot be amplified sufficiently in the amplification treatment, there arises a problem that the polymorphism is determined incorrectly. Thus, in a conventional method, with respect to a sample after being subjected to the amplification treatment, a graph of a melting curve showing the relationship between each temperature and a signal value showing the molten state of the sample or a differential value of the signal value (hereinafter referred to as “signal differential value”) is prepared, and whether or not a peak is present at around Tmm value or Tmw value is determined by visual observation. However, since specialized knowledge is required in order to make such determination in the gene analysis, it is difficult to determine easily whether what is observed is a peak or not, for example. Further, in the case of visual observation, the fact that criteria of determination vary between individuals has been perceived as a problem. Therefore, in the conventional method, the analysis has to be conducted also with respect to a sample in which nucleic acid amplification has not been conducted normally, and the labor required therefor is perceived as a problem. For such reasons, it is difficult to expand the application of a gene analysis and a gene diagnosis utilizing the melting curve analysis to the field of general analysis and diagnosis. Further, it is also difficult to analyze multiple specimens all at once from the viewpoint of its specialty or the like.    Non Patent Citation 1: Clinical Chemistry, 2000 46 (5): p. 631-635    Patent Citation 1: JP 2005-58107 A